Gap junctions are the plasma membrane specializations, present between most types of contacting cells, through which the passive movement of small molecules from one cell to another occurs (direct cell-cell communication). Many studies have indicated that cell growth, development and differentiation may be regulated by the passage of small molecules through these junctions. The long-term objective of this study is to understand the significance of gap junctional communication, how it is established and regulated. The major focus of the proposed studies is to identify the covalent modifications, such as proteolytic processing and covalent derivatization, that the gap junction undergoes along the pathway from initial synthesis of gap junction polypeptides through the assembly and regulation of mature gap junctions. Particular emphasis will be placed on how these modifications function to permit assembly of junctions and regulation of communication. The initial steps in this process will be analyzed by in vitro translation of mRNA, while latter steps will be examined in tissue culture cells, including one in which the terminal steps in assembly can be induced by incubation with cAMP. The interaction of the gap junction protein with other cellular polypeptides, including calmodulin, will be investigated. The proposed experiments capitalize on properties of recently developed antibodies to the 27,000-dalton (27kD) major polypeptide of rat liver gap junctions. These polyclonal, precipitating antibodies recognize 27kD polypeptides with similar characteristics in a wide variety of tissues and cultured cell lines. Additional antibodies, both polyclonal and monoclonal, with different specificities, will also be generated for use in this investigation. Topological studies, using both biochemical and immunochemical approaches, will be undertaken to develop a model for the disposition of the protein in the plasma membrane lipid bilayer. Attempts will be made to reconstitute gap junctional communication in vitro in both planar bilayers and liposomes to develop systems for direct analysis of the regulatory mechanisms of gap junctional communication. Information obtained from these studies ultimately will be applied to analysis of other systems, including many transformed cells in which gap junctional communication is altered or absent.